Method for treatment of indwelling catheter occlusion using fibrinolytic metalloproteinases

ABSTRACT

A method is provided for the localized intravascular administration of a fibrinolytic metalloproteinase to a human subject in amounts that are both safe and effective to lyse an occluding fibrin-containing blood clot, while also avoiding the neutralizing effects of α 2 -macroglobulin in the circulating blood. A method is also provided for the treatment of a blood clot in, around or attached to an indwelling vascular access device. A method for restoring patency and function of an indwelling vascular access device is also provided.

This application is a continuation of U.S. patent application Ser. No.10/177,916, filed Jun. 21, 2002, which is a continuation-in-part of U.S.patent application Ser. No. 09/466,276, filed Dec. 17, 1999, now U.S.Pat. No. 6,455,269, both of which are hereby incorporated in theirentireties by reference.

FIELD OF THE INVENTION

This invention relates to the therapeutic administration of fibrinolyticmetalloproteinases, and more specifically to a method for administeringsuch agents in vivo via localized delivery to vascular thrombi in orderto effect clot lysis. This invention also relates to a method for thedissolution of blood clots in an indwelling catheter, shunt or othervascular access device in order to restore function to the vascularaccess device.

BACKGROUND OF THE INVENTION

Vascular occlusions caused by blood clots such as thrombi and embolismsare serious medical maladies that can become limb or life threatening ifnot timely treated. Devices and methods have been developed for thetreatment and removal of vascular blood clots. By way of illustration,see U.S. Pat. No. 4,447,236 (Quinn), issued May 8, 1984; U.S. Pat. No.4,692,139 (Stiles), issued Sep. 8, 1987; U.S. Pat. No. 4,755,167(Thistle et al.), issued Jul. 5, 1988; U.S. Pat. No. 5,167,628 (Boyles),issued Dec. 1, 1992; U.S. Pat. No. 5,222,941 (Don Michael), issued Jun.29, 1993; U.S. Pat. No. 5,250,034 (Appling et al.), issued Oct. 5, 1993:U.S. Pat. No. 5,370,653 (Cragg), issued Dec. 6, 1994; U.S. Pat. No.5,380,273 (Dubrul et al.), issued Jan. 10, 1995; U.S. Pat. No. 5,498,236(Dubrul et al.), issued Mar. 12, 1996; U.S. Pat. No. 5,626,564 (Zhan etal.), issued May 6, 1997; U.S. Pat. No. 5,709,676 (Alt), issued Jan. 20,1998; U.S. Pat. No. 5,865,178 (Yock), issued Feb. 2, 1999, and WO90/07352 (published Jul. 12, 1990). Such methods and devices includeinfusion catheters for delivering thrombolytic or fibrinolytic agents tothe blood clot and dissolving it. Infusion catheters are typically usedin conjunction with enzymatically active agents that are capable ofdegrading the fibrin in the clot and thus effectively dissolving theclot. Such enzymes are typically referred to as “thrombolytic” or“fibrinolytic” agents.

Fibrolase is a known fibrinolytic zinc metalloproteinase that was firstisolated from the venom of the southern copperhead snake (Agkistrodoncontortrix contortrix). See Guan et al., Archives of Biochemistry andBiophysics, Volume 289, Number 2, pages 197-207 (1991); Randolph et al.,Protein Science, Cambridge University Press (1992), pages 590-600;European Patent Application No. 0 323 722 (Valenzuela et al.), publishedJul. 12, 1989; and U.S. Pat. No. 4,610,879 (Markland et al.), issuedSep. 9, 1986. Fibrolase has been shown to be fibrinolytic, and thismetalloproteinase has been documented to have proteolytic activityagainst the fibrinogen Aα-chain, with reduced proteolytic cleavage ofthe Bβ-chain and no activity against the γ-chain of fibrinogen; Ahmed etal., Haemostasis, Volume 20, pages 147-154 (1990). Because fibrin is aprincipal component of blood clots, the fibrinolytic properties offibrolase point to its potential as a clot dissolving agent for in vivothrombolytic use; see Markland et al., Circulation, Volume 9, Number 5,pages 2448-2456 (1994), and Ahmed et al., above.

Novel Acting Thrombolytic (NAT) is a modified form of fibrolase thatdiffers from fibrolase in that NAT contains 201 amino acids with anN-terminal sequence of SFPQR, whereas the N-terminal sequence of nativefibrolase begins with EQRFPQR and is 203 amino acids in length. Theamino-terminal modification was designed to prevent chemical reactionsat amino acid residues that were capable of forming a variable quantityof cyclized glutamine (pyroglutamic acid) which have the potential tocreate lot-to-lot variations in quality and uniformity of the product.Thus, NAT can be viewed as a more stable molecule.

Despite these structural differences, NAT and fibrolase are similar withrespect to enzymatic (fibrinolytic) activity. This similarity inbiological activity is consistent with data indicating that the activesite of the fibrolase molecule spans amino acids 139-159, as describedby Manning in Toxicon, Volume 33, pages 1189-1200 (1995), and itspredicted location in three-dimensional space is distant from theamino-terminus. The active site of the fibrolase and NAT moleculescontains a zinc atom which is complexed by three histidine residues.

Published literature on venom-derived fibrolase has demonstrated itsproteolytic activity against fibrinogen at the Lys⁴¹³-Leu⁴¹⁴ site andagainst the oxidized β-chain of insulin at the Ala¹⁴-Leu¹⁵ site; Retziosand Markland, Thrombosis Research, Volume 74, pages 355-367 (1994);Pretzer et al., Pharmaceutical Research, Volume 8, pages 1103-1112(1991), and Pretzer et al., Pharmaceutical Research, Volume 9, pages870-877 (1992). NAT has also been determined to have proteolyticactivity on these substrates at the same cleavage sites.

In contrast to fibrinolytic metalloproteinases such as fibrolase andNAT, clot lysing agents such as streptokinase, urokinase and tissue-typeplasminogen activator (tPA) are plasminogen activators which promotethrombolysis by activation of the endogenous fibrinolytic system. Morespecifically, plasminogen activators catalyze the conversion ofplasminogen into plasmin, a serine protease. Plasmin is capable ofcleaving fibrinogen and fibrin at arginyl-lysyl bonds, and it is throughthe generation of plasmin that the plasminogen activators ultimatelyaffect fibrin degradation and clot lysis. Current commercially availablethrombolytic agents are plasminogen activators, such as urokinase,streptokinase or tPA.

Unlike the plasminogen activator class of thrombolytic drugs,fibrinolytic metalloproteinases, such as fibrolase and NAT, do not relyon the endogenous fibrinolytic system (conversion of plasminogen toplasmin). Hence, this class of clot lysing agents can be distinguishedfrom the plasminogen activators by their unique mode of action and aredefined as “direct” fibrinolytic agents.

Alphα₂-macroglobulin is a prevalent proteinase inhibitor present inmammalian serum and is one of the largest of the serum proteins (havinga molecular weight of 725 kilodaltons). The specificity ofα₂-macroglobulin for proteinases is broad, encompassing serine,cysteine, aspartic and metalloproteinase classes. The α₂-macroglobulinmolecule is a tetramer of identical subunits that are disulfide bondedin pairs with a non-covalent association of the half molecules. Thus,under reducing conditions, native α₂-macroglobulin can be dissociatedinto its four monomeric subunits.

Each subunit of α₂-macroglobulin possesses a region that is verysusceptible to proteolytic cleavage (the “bait” region). Proteolysis ofthe bait region induces a conformational change in α₂-macroglobulin,which entraps the proteinase within the α₂-macroglobulin molecularstructure. This process is described in the literature as a “venusfly-trap” interaction. Once the proteinase is entrapped, it issterically hindered and therefore cannot access its macromolecularsubstrate.

In addition, a covalent bond can form between α₂-macroglobulin and manyof the proteinases that it entraps. As mentioned, entrapment of aproteinase induces a conformational change in the α₂-macroglobulinmolecule. It is presumed that upon this conformational change, athioester bond on the interior of the α₂-macroglobulin molecule becomesreactive and can form a covalent bond with nucleophilic residues (suchas lysine) of the entrapped proteinase. Thus, within the generalcirculation, α₂-macroglobulin can effectively neutralize a variety ofproteinases.

Moreover, the conformational change in α₂-macroglobulin brought about bythe entrapment of a proteinase results in a form that is recognized bythe reticuloendothelial system. Clearance of α₂-macroglobulin-entrappedproteinases is generally described with half-life values in minutes andis believed to occur through the low-density lipoprotein (LDL)-receptorrelated protein expressed on macrophages, hepatocytes and fibroblasts.For more on α₂-macroglobulin, see Methods in Enzymology, edited by A. J.Barrett, Academic Press, Inc., Philadelphia, (1981), pages 737-754.

Alphα₂-macroglobulin is capable of forming a macromolecular complex withfibrolase, NAT and other proteinases. Unlike some proteinases that canform a dissociable complex with α₂-macroglobulin, fibrolase and NAT aretwo examples of fibrinolytic metalloproteinases that form a complexwhich cannot be dissociated from α₂-macroglobulin under physiologicconditions. When purified human α₂-macroglobulin and NAT, for instance,are incubated together, formation of the complex begins in seconds andis nearly complete within a few minutes.

This phenomenon shows that in vitro complex formation can be rapid andis suggestive of the potential rapidity of complex formation betweenα₂-macroglobulin and NAT or other fibrinolytic metalloproteinases invivo.

Although α₂-macroglobulin is one of the major plasma proteins, there isnonetheless a finite quantity of α₂-macroglobulin in the circulationthat would be available to bind and neutralize a fibrinolyticmetalloproteinase. The α₂-macroglobulin binding capacity is thereforesaturable. Once the α₂-macroglobulin binding capacity has been exceeded,the concentration of unbound fibrinolytic metalloproteinase risesproportionally as additional fibrinolytic metalloproteinase is added tothe sample.

The presence of α₂-macroglobulin in the general circulation of a patientpresents a challenge for the systemic (for example, intravenous)administration of fibrolase, NAT and other fibrinolyticmetalloproteinases that are bound up by α₂-macroglobulin in the generalblood circulation. Unless the saturable level of innate α₂-macroglobulinis exceeded by the systemically administered dose of such fibrinolyticmetalloproteinases, the latter will effectively be neutralized andrendered ineffective for therapeutic purposes.

In one in vivo study, conducted in rabbits, the biological effectivenessof venom-derived fibrolase was examined following systemic intravenousadministration. Ahmed et al., Haemostasis, above. The dose of fibrolaseused was 3.7 milligrams per kilogram, which was estimated to yield afinal blood concentration of approximately 60 micrograms per milliliterin a 3.0-kilogram rabbit. This amount was chosen based on studiesexamining the inactivation of the enzyme in the presence of blood orplasma, presumably due to α₂-macroglobulin (see pages 336 and 339).

In another in vivo study, the biological effect of recombinant fibrolaseon clot lysis was examined in canines. Markland et al., Circulation,above. Four milligrams of this material per kilogram (of animal bodyweight) was infused over five minutes proximal to a pre-induced thrombusin the left carotid artery via a catheter device (see page 2450). Hereagain, it was noted that inactivation of fibrolase occurs in the generalblood circulation presumably due to the presence of α₂-macroglobulin(see page 2454, second column, last paragraph).

As these two studies show, the deactivating effects of α₂-macroglobulincan be overcome by either administration or systemic dosages offibrinolytic metalloproteinase that exceed the saturable level of innateα₂-macroglobulin (the rabbit study) or by delivering the enzyme locallyto the site of the clot (the dog study) and avoiding systemicadministration. On the other hand, doses of the fibrinolyticmetalloproteinase in excess of the saturable level of α₂-macroglobulin,whether delivered systemically or locally, may exceed levels that aresafe and well tolerated by the subject being treated. Notably,fibrinolytic metalloproteinases are capable of destroying not onlyfibrin, but they may also degrade other structural proteins and aretherefore potentially toxic in vivo when present in large amounts thatexceed the saturable level of α₂-macroglobulin.

The formation of a blood clot or other fibrin-based occlusion is also aconcern when using an indwelling catheter or other vascular accessdevice. There are many conditions that require recurrent or prolongeduse of a vascular access device, such as a catheter, access graft,sheath, needle, arteriovenous fistula or shunt. For example, variousprocedures associated with hemodialysis, chemotherapy, blood infusion orexchange and other procedures involving recurrent intravenous orintraartieral drug delivery (or fluid withdrawal) may involve the use ofindwelling catheter or other permanent or semi-permanent implantedmedical device. Blood clots may form in or around, or attach to, anydevice that has been introduced into a vascular space, particularlywhere the device remains in the vascular space for an extended period oftime or where the device has one or more very small openings. Inaddition, other fibrin-based occlusions may attach to any portion of anindwelling vascular access device which could prevent or hinder theproper functioning of the device.

It would be useful to have a fast and efficient method to treat, i.e.,destroy, dissolve or lyse clots or other occlusions that have formed in,around or attached to, an indwelling vascular access device. In theabsence of an efficient method to treat clots that have formed in oraround a vascular access device, the indwelling device may have to bereplaced by a physician, incurring additional cost and risk to thepatient.

Accordingly, it is an object of the present invention to provide a safeand effective method for treatment of a blood clot or occlusion in oraround an indwelling vascular access device.

It is also an object of the present invention to provide a method forrestoring patency to a fully or partially occluded indwelling vascularaccess device.

It is a further object of the present invention to provide a method forrestoring function to an indwelling vascular access device wherefunction has been altered by a fibrin-based occlusion.

It is also an object of the present invention to provide a safe andbiologically effective way of using locally administered fibrinolyticmetalloproteinases to lyse blood clots in vivo.

SUMMARY OF THE INVENTION

In certain embodiments, this invention is a method for the therapeutictreatment of a blood clot in an indwelling vascular access device byadministration through said vascular access device of a quantity offibrinolytic metalloproteinase in a pharmaceutically acceptablesolution, wherein the quantity does not exceed 1.7 milligrams offibrinolytic metalloproteinase per kilogram of body weight of the humansubject being treated. In certain embodiments, the vascular accessdevice is indwelling in an artery or vein of a human subject. In certainembodiments, the indwelling vascular access device is a catheter, shunt,access graft, needle or sheath. In certain embodiments, the vascularaccess device is employed to introduce or remove fluids from a vasculararea. In certain embodiments, the vascular access device of thisinvention is used in connection with hemodialysis, blood transfusion,removal or exchange, chemotherapy or any other procedure that requiresthe introduction or removal of fluids from a vein or artery.

In certain embodiments, this invention is a method for lysing bloodclots in an indwelling vascular access device by local administration tosaid clot of a quantity of fibrinolytic metalloproteinase that complexeswith alpha-two macroglobulin in an amount sufficient to facilitatelysing of the clot while not exceeding a level significantly abovesaturable level of alpha-two macroglobulin in said human subject, in apharmaceutically acceptable solution.

In certain embodiments, this invention is a method for restoring patencyto a fully or partially occluded indwelling vascular access device. Incertain embodiments, this invention is a method for restoring functionto an indwelling vascular access device.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1C. FIG. 1A is a baseline angiogram of the carotid artery in anadult pig prior to balloon catheter induced-injury and the formation ofan occlusive thrombus FIG. 1B is an angiogram taken on day 4 in the sameanimal, prior to the administration of NAT in accordance with the methodof this invention. FIG. 1C is an angiogram at 2 hours followingadministration of 30 mg of NAT through a “PRO” catheter (see FIG. 3 foran illustration of this device).

FIG. 2 illustrates a type of catheter device designed for localizeddelivery of a thrombolytic agent in a blood vessel.

FIG. 3 is a side cross-sectional view of an alternate type of catheterdevice for localized delivery of a thrombolytic agent in a blood vessel.

FIG. 4 is a histogram of estimated maximum dose of NAT in patients withperipheral vascular occlusion.

FIG. 5 illustrates the use of NAT in an in vitro catheter occlusionmodel.

FIG. 6 illustrates summary results of numerous experiments, theresulting mean data showing that NAT resolves in vitro occlusions up to40% faster than Abbokinase® Open Cath®, a commercial urokinase productfor catheter clearance.

DETAILED DESCRIPTION OF THE INVENTION

In certain embodiments this invention is a method for the treatment of ablood clot in vivo, in human subjects, by a fibrinolyticmetalloproteinase, comprising locally administering a safe, biologicallyeffective amount of the fibrinolytic metalloproteinase to the bloodclot, such as by use of a catheter or other vascular access device.Typically, stationary fibrin clots will be located in a blood vessel(arterial or venous, native or synthetic (e.g., a graft)) in a humansubject. In certain embodiments, this invention is a method for treatinga blood clot located in, around or attached to an indwelling catheter,shunt, needle or other vascular access device. In certain embodiments,this invention is a method for restoring patency to a fully or partiallyoccluded indwelling vascular access device. In certain embodiments, thisinvention is a method for restoring function to a vascular access devicewhere function has been altered or compromised to some extent by afibrin-based occlusion.

By “safe, biologically effective” amount is meant an amount sufficientto degrade fibrin and facilitate lysing of the clot (i.e., thrombus),while not exceeding levels significantly above the saturable level ofα₂-macroglobulin in the circulatory system of the patient being treated(i.e., levels that may cause damage to blood vessel walls). Typically,this amount will be in the range between 0.025 and 1.7 milligrams perkilogram of body weight for the human subject being treated, asdetermined from a study conducted with blood samples from human subjectsthat have been studied for in vitro α₂-macroglobulin content and bindingcapacity. From the in vitro results of this study, it has been possibleto define the saturable level in vivo of α₂-macroglobulin for allpractical purposes, thus enabling the delineation of a biologicallyeffective amount that takes into account not only the minimum level offibrinolytic metalloproteinase required for biological effectiveness,but also the maximum level for well-tolerated administration. This studyis described in detail further below among the Examples.

The terms “locally” or “localized” as applied to the manner of deliveryof the fibrinolytic metalloproteinase herein refers to intra-arterial orintravenous administration either directly to the blood clot itself(i.e., intrathrombus) or in close proximity to the clot (either proximalor distal relative to blood flow) and near enough for the majority ofthe fibrinolytic metalloproteinase to be absorbed by the clot. For thetreatment of clots in, around or attached to a vascular access device,or to restore patency or function to vascular access device, delivery ofa fibrinolytic metalloproteinase is generally effected via the vascularaccess device itself, and is therefore inherently local. In certainembodiments, a secondary vascular access device, such as a catheter, maybe used to deliver a fibrinolytic metalloproteinase to an implantedmedical device, such as a stent.

The term “catheter delivery means” or “vascular access device” isemployed herein in the conventional sense of referring to a tubularmedical device for insertion into canals, vessels, passageways or bodycavities for the purpose of injecting or withdrawing fluids or to keep apassageway open. In general, such means will typically comprise anelongated flexible catheter body containing one or more interiorpassageways (or “lumens”); a proximal portion which allows material(i.e., clot lysing agent) to be introduced into the catheter body and toflow through the lumen; a distal portion optionally having a taperedend; and one or more exit ports at or near the end of the distal portionto permit material to exit the catheter in response to applied pressure.

The terms “proteolytic degradation,” dissolution,” “lysis” and“therapeutic treatment” are all used herein to refer to the degradation,disintegration, decomposition, break up or other removal of a blood clotor other fibrin-based occlusion. The blood clot may partially or totallyocclude the lumen of a vessel or medical device. Similarly, “restoringpatency” refers to any measurable increase in blood flow (for example,by volume or speed) through a vessel or device lumen or exit port due tothe dissolution of a blood clot therein.

The term “blood clot” or “occlusion” as used herein refers to anyfibrin-based mass, cluster, obstruction or growth. Typically blood clotsresult when blood coagulates in an artery or vein and impede, obstruct,hinder or block, totally or partially, the flow of blood therethrough.Blood clots can also be formed in or around any foreign objectintroduced into a vascular space. Blood clots can be stationary, such asa thrombus (a blood clot that forms in a vessel and remains there) or itmay be transportable, such as an embolism (blood clots that travels fromthe site where it formed to another location in the body). Blood clotscan also vary widely in size, shape and composition, and can includespherical masses as well as flaps or other planar structures. Sometimes,a piece of atherosclerotic plaque, small pieces of tumor, fat globules,air, amniotic fluid or other materials found in the blood can act in thesame manner as a blood clot. To the extent such masses contain fibrin orother substances susceptible to degradation by a fibrinolyticmetalloproteinase, one method of the present invention would be usefulfor the treatment thereof.

Another method of this invention is illustrated further below withrespect to peripheral arterial occlusion (PAO). PAO finds its origins inperipheral vascular disease due to atherosclerosis. The symptoms developslowly over many years as atherosclerosis progresses, with a criticalischemic level being reached in about 15 to 20% of patients with lowerextremity disease. Medical therapy is limited and predominantly aimed atprevention or risk reduction using medications such as lipid-lowering orantiplatelet agents, smoke cessation programs and physical exercise.Jackson and Clagett, Chest, Volume 114, pages 666S-682S (1998).

The clinical manifestations of peripheral vascular disease may includeacute occurrences of limb-threatening ischemia or the presence of morechronic evidence of vascular disease (i.e., intermittent claudications).Outside of the aforementioned preventive measures, chronic PAO istypically not treated until the onset of severe lifestyle limitation orlimb-threatening ischemia. Depending on the vessel segment affected andthe extent of occlusion, available medical interventions includepercutaneous transluminal angioplasty, surgical revascularization, andthrombolysis. Studies have shown that the intra-arterial infusion ofclot lysing agents, particularly in the early stages of occlusion, canavoid the need for surgical intervention. As demonstrated in theRochester trial, which compared thrombolysis with the plaminogenactivator urokinase to surgery in the treatment of acute PAO (Ouriel etal., Journal of Vascular Surgery, 1994, Volume 19, pages 1021-1030),approximately 33 percent of patients in the thrombolysis arm of thestudy were successfully treated with medical intervention only, therebyavoiding a more invasive procedure. In contrast, 98 percent of patientsin the operating arm were subjected to an endovascular or surgicalprocedure.

Other medical disorders involving occlusive blood clots can beeffectively treated in a similar manner by the present method,including, but not limited to, acute myocardial infarction, ischemicstroke, deep venous thrombosis and pulmonary embolism. In certainembodiments, the method of this invention can also be employed todissolve clots which occur with chronically implanted medical devicessuch as indwelling catheters and hemodialysis access grafts. Otherexemplary implanted medical devices include shunts, access parts,needles, sheaths or other vascular access devices.

In certain embodiments, this invention is applicable for the therapeuticdelivery of any fibrinolytic metalloproteinase that is capable of beingcomplexed with α₂-macroglobulin. Such fibrinolytic metalloproteinases,if naturally occurring, may be purified from their natural sources,e.g., fibrolase from snake venom. Alternatively, polypeptidefibrinolytic metalloproteinase agents the nucleic acid and amino acidsequences of which are known may be produced by utilizing conventionalmethods of recombinant expression and purification.

In general, recombinant methods employ a DNA molecule encoding thefibrinolytic metalloproteinase of interest which is inserted into anappropriate vector for expression in a suitable host cell. The vector isselected to be functional in the particular host cell employed, i.e., iscompatible with the host cell machinery, such that expression of the DNAcan occur. The vectors may also contain a 5′ flanking sequence (alsoreferred to as a “promoter”) and other expression regulatory elementsoperatively linked to the DNA to be expressed, as well as other knownelements, such as an origin of replication element, a transcriptionaltermination element, a complete intron sequence containing a donor andacceptor splice site, a signal peptide sequence, a ribosome binding siteelement, a polyadenylation sequence, a polylinker region for insertingthe encoding nucleic acid, and a selectable marker element. The vectormay also optionally contain a “tag” sequence, i.e., an oligonucleotidesequence located at the 5′ or 3′ end of the polypeptide-coding sequencethat encodes polyHis or another small immunogenic sequence. This tagwill be expressed along with protein of interest, and can serve as anaffinity tag for purification of this polypeptide from the host cell. Ifdesired, the tag can subsequently be removed from the purifiedpolypeptide by various means, for example, with use of a selectivepeptidase.

In those cases where it is desirable for the polypeptide to be secretedfrom the host cell, a signal sequence may be used to direct thepolypeptide out of the host cell where it is synthesized. Typically, thesignal sequence is positioned in the coding region of nucleic acidsequence, or directly at the 5′ end of the coding region. Many signalsequences have been identified, and any which are functional in theselected host cell may be used.

After the vector has been constructed and a nucleic acid has beeninserted into the proper site of the vector, the completed vector may beinserted into a suitable host cell for amplification and/or polypeptideexpression. Host cells may be prokaryotic (such as E. coli) oreukaryotic (such as a yeast cell, an insect cell, or a vertebrate cell).

Suitable host cells or cell lines may be mammalian cells, such asChinese hamster ovary cells (CHO) or 3T3 cells. The selection ofsuitable mammalian host cells and methods for transformation, culture,amplification, screening and product production and purification areknown in the art. Other suitable mammalian cell lines are the monkeyCOS-1 and COS-7 cell lines, and the CV-1 cell line. Further exemplarymammalian host cells include primate cell lines and rodent cell lines,including transformed cell lines. Normal diploid cells, cell strainsderived from in vitro culture of primary tissue, as well as primaryexplants, are also suitable. Candidate cells may be genotypicallydeficient in the selection gene, or may contain a dominantly actingselection gene. Still other suitable mammalian cell lines include butare not limited to, HeLa, mouse L-929 cells, 3T3 lines derived fromSwiss, Balb-c or NIH mice, BHK or HaK hamster cell lines.

Also useful as host cells are bacterial cells, for example, variousstrains of E. coli, and various strains of yeast cells.

Insertion (also referred to as “transformation” or “transfection”) ofthe vector into the selected host cell may be accomplished using suchmethods as calcium phosphate, electroporation, microinjection,lipofection or the DEAE-dextran method. The method selected will in partbe a function of the type of host cell to be used. These methods andother suitable methods are well known to the skilled artisan.

The host cell, when cultured under appropriate conditions, cansynthesize the fibrinolytic metalloproteinase of interest. The hostcells may be cultured using standard media well known to the skilledartisan. The media will usually contain all nutrients necessary for thegrowth and survival of the cells. Suitable media for culturing E. colicells are, for example, Luria Broth (LB) and/or Terrific Broth (TB).Suitable media for culturing eukaryotic cells are RPMI 1640, MEM, DMEM,all of which may be supplemented with serum and/or growth factors asrequired by the particular cell line being cultured.

Typically, an antibiotic or other compound useful for selective growthof the transformed cells only is added as a supplement to the media. Thecompound to be used will be dictated by the selectable marker elementpresent on the plasmid with which the host cell was transformed. Forexample, where the selectable marker element is kanamycin resistance,the compound added to the culture medium will be kanamycin.

The amount of protein produced in the host cell can be evaluated usingstandard methods known in the art, including Western blot analysis,SDS-polyacrylamide gel electrophoresis, non-denaturing gelelectrophoresis, HPLC separation, immunoprecipitation, and/or activityassays such as DNA binding gel shift assays.

If the protein is secreted from the host cells other than gram-negativebacteria, the majority will likely be found in the cell culture medium.If it is not secreted, it will be present in the cytoplasm. Forintracellular protein, the host cells are typically first disruptedmechanically. For protein having a periplasmic location, eithermechanical disruption or osmotic treatment can be used to release theperiplasmic contents into a buffered solution, and the polypeptide isthen isolated from this solution. Purification from solution canthereafter be accomplished using a variety of techniques.

If the protein has been synthesized so that it contains a tag such ashexahistidine or other small peptide at either its carboxyl or aminoterminus, it may essentially be purified in a one-step process bypassing the solution through an affinity column where the column matrixhas a high affinity for the tag or for the polypeptide directly (i.e., amonoclonal antibody). Where the polypeptide has no tag and no antibodiesare available, other well known procedures for purification can be used,for example, ion exchange chromatography, molecular sievechromatography, reversed phase chromatography, HPLC, native gelelectrophoresis in combination with gel elution, and preparativeisoelectric focusing (“Isoprime” machine/technique, Hoefer Scientific).In some cases, two or more of these techniques may be combined toachieve increased purity.

Novel Acting Thrombolytic (NAT) polypeptide utilized herein toillustrate the practice of this invention refers in general to thefibrinolytically active metalloproteinase of SEQ ID NO: 1. The NATpolypeptide is encoded by the cDNA molecule of SEQ ID NO: 2, althoughany DNA molecule of variant sequence encoding the same polypeptide maybe used for expression and manufacture in accordance with specificmethods which are referred to further below.

Fibrolase has been described in the scientific and patent literature;see references above. Typically, the form of fibrolase which is employedin the practice of this invention will be of SEQ ID NO: 3, which isencoded by the cDNA molecule of SEQ ID NO: 4 or variants thereofencoding the same amino acid sequence.

Preferably, a yeast expression system is employed for recombinantexpression of NAT. Special mention is made of Pichia strains, forexample, Pichia pastoris, as being the most advantageous and preferredfor use. A detailed description of such a system may be found in U.S.Pat. No. 4,855,231 (Stroman et al.), U.S. Pat. No. 4,812,405 (Lair etal.), U.S. Pat. No. 4,818,700 (Cregg et al.), U.S. Pat. No. 4,885,242(Cregg), and U.S. Pat. No. 4,837,148 (Cregg), the disclosures of whichare hereby incorporated by reference. Expression of fibrolase in such asystem will typically involve a DNA molecule of SEQ ID NO: 5, whichencodes “prepro” sequence (nucleotides 1-783) in addition to the“mature” polypeptide (nucleotides 784-1392). The expression of NAT insuch a system will typically involve a DNA molecule of SEQ ID NO: 6,which encodes “prepro” sequence (nucleotides 1-783) in addition to the“mature” polypeptide (nucleotides 784-1386).

The fibrinolytic metalloproteinase employed in accordance with thisinvention, whether it be NAT, fibrolase, or some other fibrinolyticmetallo-proteinase, is administered in the form of a pharmaceuticallyacceptable solution, alone or containing additional pharmaceuticallyacceptable ingredients. If desired, such solutions may comprise, inaddition to the fibrinolytic metalloproteinase and a solvent (i.e.,distilled water or physiological saline), standard ingredients such asstabilizers (to prevent protein aggregation or physical or chemicaldegradation in aqueous media), bulking agents (to provide bulk),diluents, antibacterial agents, viscosity adjusting agents,anti-oxidants, and so forth, in conventional amounts.

Known excipients which can be included in the formulation includepolyols (including mannitol, sorbitol and glycerol); sugars (includingglucose and sucrose); and amino acids (including alanine, glycine andglutamic acid). See, for example, Remington's Pharmaceutical Sciences,Mack Publishing Company, Easton, Pa. See also WO 01/24817 A2 (theentirety of which is herein incorporated by reference) which disclosesvarious compositions of fibrinolytic agents useful in the presentinvention.

Desirably, the pharmaceutical composition will be buffered (with abiocompatible buffering agent, for example, citric acid or citric acidsalt) to a pH which is at or near neutral (7.0) prior to administration,and usually between about 6.5 and about 8.0 pH (±0.5).

If the metal ion of the fibrinolytic metalloproteinase is zinc, such aswith fibrolase or NAT, it may be preferable to include a water-solublezinc salt (for example, zinc sulfate or zinc acetate) as a stabilizer.To further enhance the long-term stability and shelf life of thecomposition, it may also be advantageous to freeze the solution or toconvert it to a lyophilized (freeze-dried) product which is thawed orreconstituted prior to use, as the case may be.

By way of illustration, a freezable liquid medicinal composition whichmay be employed in the method of this invention comprises fibrolase orNAT, a water soluble zinc salt, a citric acid buffer, optionally anadditional stabilizer selected from the group consisting of watersoluble calcium salts, and optionally a bulking agent (for example,mannitol). A surfactant, such as Tween® 80 (BASF, Gurnee, Ill.), mayalso be added to increase freeze-thaw stability. Tris buffer (Sigma, St.Louis, Mo.) or another buffer with a buffer capacity above pH 7.0 may beadded to stabilize the pH at or above pH 7.4. Most of these ingredientswill be present in minor amounts ranging from 0.001 to 2.0 millimolar(mM) or less than ten percent (w/v). The buffering agent will be addedin an amount sufficient to achieve the desired pH, and this amount mayvary depending on the specific formulation.

By way of further illustration, a lyophilizable or lyophilizedpharmaceutical composition which can be used in the method of thisinvention comprises fibrolase or NAT, a zinc stabilizer (e.g., watersoluble zinc salt such as the above), and a citric acid buffer, with orwithout other excipients (e.g., bulking agent such as mannitol, glycine,or the like). The lyophilized composition may also contain adisaccharide sugar, such as sucrose or trehalose, as a lyoprotectant.

A surfactant, such as Tween 80®, may be added to protect againstlyophilization stresses on the fibrinolytic metalloproteinase (e.g.,fibrolase or NAT). The pH will ideally be maintained at pH 8.0±0.5,using a suitable buffer with a pK_(a) in this range (for example, Tris).Amounts of ingredients will be in accordance with the above.

As mentioned, in certain embodiments, the method of this invention isemployed to locally administer biologically effective amounts of afibrinolytic metalloproteinase that are in the dose range between 0.025and 1.7 mg/kg. Preferably, this amount will in the range from about 0.1to about 0.5 mg/kg. Solution strengths will be formulated accordingly,with dilution to be affected as needed upon administration.

In contrast to treatment of thrombosis in a native artery or vein, suchas PAO, treatment of a blood clot occurring in an implanted orindwelling vascular access device presents addition challenges. Forexample, in the case of an occluded central venous line, the “deadvolume” of the catheter (i.e., the inner diameter of catheter multipliedby its length) defines the maximum volume that can be contained withinthe catheter. For example, a catheter with a 1.0 mm internal diameterand a 40.0 cm length possesses a dead volume of ˜0.3 ml. Because volumecan be so limited, it is important to define the proper solutionstrength. Accordingly, increasing solution strength or concentration isan effective means for increasing the amount of drug available todissolve a target blood clot.

In certain embodiments, the method of the present invention concerns theuse of a fibrinolytic metalloproteinase for the treatment of occlusionsassociated with a vascular access device. Such clots can occur in,around or be attached to, a vascular access devices. Typically, clotswill form in the lumen or exit port of a device such as a catheter, andmay interfere with the function of the catheter. Clots can also occur onthe exterior of a vascular access device, such as a flap that may coveran exit port and thereby prevent the introduction of a drug or removalof blood. Clots can also attach to a vascular access device, interferingwith the proper functioning of the device. The interference can resultfrom a partial occlusion or blockage, i.e., some fluid may pass through,or a total occlusion in which no fluid can pass.

To demonstrate one method of the present invention, an in vitro catheterocclusion model was used to simulate a clot in an indwellingcatheter-type device. In this model, collagen-coated Pasteur pipetteswere used to simulate an indwelling portion of a catheter. Human bloodwas drawn via a finger stick by capillary action to a length of 3.0 mmand allowed to clot. The tip of one pipette was introduced into a vialcontaining saline and another into a 4 mg of NAT (100 ul of a 40 mg/mlsolution strength) solution of NAT. FIG. 5 illustrates the results ofthis in vitro catheter occlusion model, which indicate that the clot inthe saline is still intact, while the clot in 40 mg/ml is active indissolving the clot.

In another experiment, a separate group of pipettes (each containing ablood clot at its tip) was introduced into and incubated in a vialcontaining the following: 100 ul of saline, 100 ul of a urokinasesolution (5000 IU/ml) and 100 ul of a NAT solution where the quantity ofdrug was varied from 0.5 to 4 mg by increasing the solution strengthfrom 5 mg/ml to 40 mg/ml. FIG. 6 illustrates summary results of numerousexperiments, the resulting mean data showing that NAT resolves in vitroocclusions up to 40% faster than Abbokinase® Open Cath®, a commercialurokinase product for catheter clearance (second column, UK 500 I.U.).FIG. 6 also demonstrates the dose-dependent nature of NAT in terms ofthe speed at which NAT acts to proteolytically degrade a blood clot.

For treatment of a relatively small indwelling catheter occlusion, suchas that simulated by the in vitro catheter occlusion model, a preferredrange for treatment of would be approximately 5 to 40 mg/ml offibrolase, NAT or other fibrinolytic metalloproteinase. However,depending on the type and size of the vascular access or other medicaldevice, and the position, size and extent of the clot or occlusion, muchsmaller or larger doses may be required. For example, a clot in arelatively large hemodialysis shunt would likely require a higherconcentration of fibrinolytic metalloproteinase than a clot blocking theexit port of a 1.0 mm diameter catheter. Accordingly, concentrations forthis indication may range anywhere from 0.1 mg/ml or less, to over 80mg/ml.

For blood clots located in, around or attached to an indwelling vascularaccess device, an effective dose can be delivered in any number of ways,including pulsatile infusion, continuous infusion, bolus administration,or a combination of all three. Given the difficulty posed by “deadvolume” constraints, a bolus administration is generally preferred.

In the typical case, the method of this invention is carried out inconjunction with a catheter-directed thrombolysis procedure. Suchprocedures involve the use of a pre-sterilized catheter-type drugdelivery device, the side walls of which may be made of a thin,semi-rigid or flexible biocompatible material (for example, apolyolefin, fluoropolymer, or other inert polymer). In general, suitablecatheters contain at least one interior cavity (or lumen) running thelength of the device. The material from which the catheter isconstructed is flexible enough to be moved through the interior of thevasculature without causing injury to the blood vessel walls, yetsufficiently rigid to extend over a distance to the site of treatmentwhile the interior cavity of the device remains fully distended.Typically, such catheter devices will range from 2 to 20 on the Frenchscale for catheter diameters (⅓ millimeter equals 1 French) and from twoto six feet or more in length.

Exemplary catheter devices for the intravascular delivery ofthrombolytic medication in accordance with this invention areillustrated in FIGS. 2 and 3, the practical applications of which aredescribed in detail in the examples below. However, any conventionalcatheter delivery or vascular access device which is suitable for thismethod may be utilized, including but not limited to the specificdevices referred to herein.

For example, FIG. 2 illustrates a type of catheter device designed forlocalized delivery of a thrombolytic agent in a blood vessel. Thedevice, shown in side cross-sectional view, contains “side holes” at thedelivery end through which the infusate (thrombolytic agent) is emittedunder applied fluid pressure. The diameters of the catheter tubes inthis figure and the following figure relative to overall size areexaggerated to show the details better. See also FIG. 3, whichillustrates a side cross-sectional view of an alternate type of catheterdevice for localized delivery of a thrombolytic agent in a blood vessel.This device contains thin slits, referred to as “pressure responseoutlets” (PRO), which have been cut into the catheter wall at regularintervals such that infusate escapes when the fluid pressure within thecatheter reaches a critical point, causing the slit to open. The devicecan be used in conjunction with an automated, piston-driven, pulsedinfusion device (not shown, but exemplified further below) which iscapable of delivering pulses of drug infusion.

For blood clots that occur in a vein or artery and are not associatedwith the introduction of a vascular access or other medical device, aneffective dose of fibrinolytic metalloproteinase can be deliveredthrough the catheter to the local site of treatment by pulsatileinfusion, continuous infusion, bolus administration, or a combination ofall three. The solution strength (i.e., concentration) of fibrinolyticmetalloproteinase in the treatment solution is also an importantparameter for these types of clots. More specifically, a range betweenthe minimum dilution of the fibrinolytic metalloproteinase foreffectiveness at the lower end (which is especially important for bolusadministration) and the maximum solubility of the fibrinolyticmetalloproteinase at the higher end should be selected. In general,solution strengths in the range from about 0.1 to about 80 mg/ml areemployed. The volume of the bolus (or total volume of multiple bolusesin the case of a “pulsed” delivery) is then selected accordingly todeliver an effective amount of fibrinolytic metalloproteinase within theranges prescribed above.

Description of Specific Embodiments

The invention is further illustrated in the following examples, whichare meant to be illustrative only and not to limit the invention to thedescribed embodiments. In these examples, and throughout the descriptionof this invention, “kg” indicates kilograms of body weight per testsubject, “mg” indicates milligrams, “mL” indicates milliliters, and“min” indicates minutes. The fibrinolytic metalloproteinase illustrated,namely Novel Acting Thrombolytic (NAT), was recombinantly-derived andmade in accordance with methods referred to above.

EXAMPLE 1 Thrombolysis in Subacute Thrombosis of the Adult Pig CommonCarotid Artery

NAT was been studied in a model of subacute thrombosis of the carotidartery in adult pigs, averaging 75 kg in body weight, at a contractlaboratory (Charles River Laboratories, Southbridge, Mass.). The intentof this study was to establish the fibrinolytic activity of NAT in athrombosis model which is relevant to peripheral artery occlusion inhumans.

In this animal model, the carotid artery was thrombosed along its entirelength (approximately 20 centimeters from origin at the aorta to thecarotid bifurcation) by a combination of balloon injury, thrombin andstasis. The size of the thrombus approaches the size of thrombusencountered clinically in humans with peripheral arterial occlusion.After successful thrombosis, the animal was allowed to recover for aperiod of four days. A four-day period was selected to allow extensivecross-linking of fibrin, remodeling of thrombus, and infiltration ofcells. Notably, both the size and age of the thrombus in this model arereasonable representations of the size and duration of ischemic symptomsreported in the most recently published TOPAS trial of thrombolysis withplasminogen activators in peripheral arterial occlusion in humans. Forreference, see Ouriel et al., New England Journal of Medicine, Volume338, pages 1105-1111 (1998).

Briefly, the common carotid artery can be thrombosed along its entirelength by fluoroscopically directed balloon injury. The balloons thatare used are oversized and non-compliant. The balloons are inflated atpressures of up to twelve atmospheres, which causes a crushing injury tothe intimal layer of the vessel. While the balloon is inflated, it ismoved back and forth in order to strip away the vascular endothelium.

The ballooning procedure is very injurious and creates a highlythrombogenic vessel surface and is repeated throughout the entire lengthof the common carotid artery. After thoroughly injuring the entireartery, the balloon is withdrawn to a proximal position near the aortaand inflated to occlude flow of blood through the vessel. Whileoccluded, fifty Units of bovine thrombin is injected through the distalport of the balloon catheter in order to stimulate coagulation. Theballoon remains inflated for a period of thirty minutes, which resultsin thrombotic occlusion of the vessel. After thirty minutes, the balloonis deflated and an angiogram is performed to confirm that the vessel hasbecome occluded. With these procedures, occlusion of the vessel wasachieved in greater than ninety percent of cases.

FIG. 1A is a baseline angiogram of the carotid artery in an adult pigprior to balloon catheter induced-injury and the formation of anocclusive thrombus. The arrow indicates the position and the presence ofcontrast media in the left common carotid artery, indicating that bloodflow in the artery is unobstructed (i.e., the blood vessel is “patent”or has “ ”). FIG. 1B is an angiogram taken on day 4 in the same animal,prior to the administration of NAT in accordance with the method of thisinvention. The arrow indicates the position of the left common carotidartery, however, contrast media does not flow in the artery due to thepresence of an occlusive thrombus. FIG. 1C is an angiogram at 2 hoursfollowing administration of 30 mg of NAT through a “PRO” catheter (seeFIG. 3 for an illustration of this device). The arrow indicates thepresence of contrast media in the vessel, demonstrating that patency hasbeen restored in the left carotid artery. Minimal residual thrombus isvisible in the lumen of the artery.

Once thrombosed, the balloon catheter, guide catheter and access sheathare removed and the animal is allowed to recover over a period of fourdays. On the fourth day, the animals are re-anesthetized, the occlusionis reconfirmed, and a multiple side hole drug delivery catheter (seeFIGS. 2 and 3) is advanced under fluoroscopic guidance and positionedsuch that the side holes are located within the thrombus. Thrombolysiswith NAT was angiographically observed using fixed NAT dosages whichranged from 10 to 30 mg (or approximately 0.1 to 0.4 mg/kg on a weightadjusted basis), as shown in FIGS. 1A-1C.

As the image in FIG. 1B illustrates, NAT is effective in restoringantegrade flow as assessed by angiography. To be more quantitative, flowin the target vessel is qualitatively scored according to a 4-pointscale (ranging from 0 to 3) where:

-   -   0=no flow    -   1=flow estimated to be less than 30% of the contralateral        (non-thrombosed) carotid    -   2=flow estimated to be 30-80% of the contralateral carotid    -   3=flow which is indistinguishable from the contralateral carotid

The image shown in FIG. 1B was scored as flow grade equal to 3, afrequent result in thrombosed vessels treated with a fixed 30 mg dosageof NAT (approximately 0.4 mg/kg in a 75 kg pig). Tables 1-3 in thefollowing examples below illustrate the treatment regimen and mean flowscores derived from serial angiograms. In all of the studies,respiration, body temperature, heart rate and arterial blood pressureswere continually monitored and remained within physiologic ranges withno changes observed upon administration of NAT.

EXAMPLE 2 Selection of PRO Catheters and Pulse-Spray Delivery

In the clinical management of peripheral arterial occlusion,thrombolytic agents are delivered through catheters which are positionednear or embedded into the thrombus. As shown in FIGS. 2 and 3, there aretwo types of commonly used catheters.

One variety, the “side hole” catheter (FIG. 2), has tiny round sideholes (2) cut into the catheter (4) near closed distal end 6, and anentry port 8 (for the solution of fibrinolytic metalloproteinase) inmating ring 10 affixed at proximal end 12. Catheter 4 is constructed ofa flexible, elongated, biocompatible polymer tubing material which ishollow and thin-walled and has a uniform diameter of 2 to 20 French, andpreferably 3 to 5 French. The catheter contains two radiopaque markers14 on the exterior surface near distal end 6 which demarcate the portionof the catheter containing side holes 2. In practice, the catheter isinserted into a surgical opening in the occluded artery or vein and,while being observed via fluoroscopy in accordance with standardapproved procedures, is moved carefully through the blood vessel suchthat distal end 6 is positioned into or near the thrombus. Markers 14,which show up clearly on a fluoroscope image, can serve as a guide forpositioning that portion of the catheter such that infusate emergingfrom side holes 2 will contact the thrombus directly. A pharmaceuticalsolution of the fibrinolytic metalloproteinase is then injected undergentle pressure from syringe-like reservoir 16 into entry port 8 and isimpelled toward distal end 6, emerging through holes 2 into thethrombus, causing degradation of the fibrinous material.

When infusions of a fibrinolytic metalloproteinase are performed usingthis type of catheter, most of the fibrinolytic agent-containingsolution tends to escape through the proximal side holes (i.e., thosenearest to drug entry port 8), which has a negative impact in theuniformity of drug delivery at the site of treatment. There is also apossibility of backflow of blood into the catheter through side ports 2under negative pressure.

Another variety of catheter, also composed of a hollow, thin-walled,biocompatible polymer material, shown in FIG. 3, has extremely thinslits 2 that are laser cut into flexible catheter 4 at regular intervalsnear closed distal end 6. The slits, which are referred to as pressureresponse outlets (“PRO”), are tight enough that infusate will not escapeunless the fluid pressure within the catheter reaches a critical pointand causes the slits to distend simultaneously and thus opentemporarily. The catheter can also contain exterior radiopaque markers 8to assist in the positioning of the device at the site of the thrombus.

Ideally, such “PRO” infusion catheters are used in conjunction with anautomated, piston-driven, pulsed infusion device (not shown) that iscapable of delivering low volume regulated pulses of drug infusion intoentry port 10 in mating ring 12 affixed at proximal end 14 of catheter4. When a pulse is delivered, the pressure within the catheter risesmomentarily. In response, the pressure response outlets (slits 2) openmomentarily and allow the infusate (e.g., pharmaceutical solution offibrinolytic metalloproteinase) to escape. The theoretic advantage ofpulsed delivery of infusate and a PRO-type catheter is that infusate isdelivered uniformly through the slits along the entire length of thecatheter, whereas infusate delivered through the “side hole” catheter(FIG. 2) follows a path of least resistance and flows out the proximalside holes in a non-uniform manner as mentioned.

A pig model of four-day old carotid thrombosis was utilized to assessperformance of the above-mentioned two catheter types, using fixeddosages of 30 mg of NAT (approximately 0.4 mg/kg in a 75 kg pig). Theresults are summarized in Table 1. TABLE 1 Comparison of AngiographicFlow Scores Obtained with NAT Using Side Hole and PRO Infusion CathetersDELIV TIME (deliv- ANGIOGRAPHIC FLOW SCORE DRUG DOSE CATH ery) 0 30 m 1hr 2 hr 4 hr NAT 30 mg CM 60 min 0.0 ± 1.2 ± 1.6 ± 1.8 ± 2.0 ± n = 5(infu- 0.0 0.4 0.5 0.4 0.7 sion) NAT 30 mg PS 60 min 0.0 ± 1.5 ± 1.5 ±2.3 ± 2.5 ± n = 4 (0.1 mL 0.0 0.6 0.6 0.5 0.6 pulse)CM Cragg-McNamara ™ valved infusion “side hole” type catheter (MicroTherapeutics, Inc., San Clemente, California).PS “pulse-spray” delivery as defined by use of pressure response outlet(PRO) catheter (Uni*Fuse Catheter ™, AngioDynamics, Inc., Queensbury,New York) used in conjunction with automated, pulsed infusion device(PULSE*SPRAY INJECTOR Model PSI-1, AngioDynamics, Inc., Queensbury, NewYork).

As shown in Table 1, angiographic flow scores in the group treated withthe pulse-spray modality showed slightly higher initial flow scores atthe thirty-minute angiogram which appeared to persist to the four-hourtimepoint. Although statistical differences were not obtained, theangiographic results have generally been judged to be superior.Therefore, a PRO-type catheter in conjunction with pulse-spray deliveryis the preferred mode of delivery for a fibrinolytic metalloproteinasein accordance with this invention.

EXAMPLE 3 Assessment of Drug Delivery Time

Acute peripheral arterial occlusion is typically treated withplasminogen activators, such as urokinase, delivered as an infusionwhich is often twenty-four hours in duration, and occasionally as longas forty-eight hours. The lengthy infusion is presumed to maintain a lowlevel of plasmin generation over a prolonged period of time in order toeffectively dissolve the occlusive thrombus. As both NAT and fibrolaseare fibrinolytic metalloproteinases, such prolonged infusions may not benecessary. To assess whether the delivery rate affects angiographic clotlysis, a fixed 30 mg dose of NAT (roughly 0.4 mg/kg in a 75 kg pig) wasdelivered using PRO catheters and the pulse spray device. Using 0.1 mLpulse volumes, a 5 mg/mL NAT solution was delivered over six minutes(ten pulses per minute) or over sixty minutes (one pulse per minute).The results are shown in Table 2. TABLE 2 Comparison of Drug DeliveryTimes for NAT Delivered by PRO Catheter and Pulse-Spray Injector DELIVTIME (pulse ANGIOGRAPHIC FLOW SCORE DRUG DOSE CATH volume) 0 30 m 1 hr 2hr 4 hr NAT 30 mg PS 6 min 0.0 ± 2.7 ± 2.7 ± 2.7 ± 2.7 ± n = 3 (0.1 0.00.6 0.6 0.6 0.6 mL) NAT 30 mg PS 60 min 0.0 ± 0.0 ± 1.0 ± 1.3 ± 1.7 ± n= 3 (0.1 0.0 0.0 1.0 1.2 0.6 mL)

As shown in Table 2, the delivery of 30 mg NAT over six minutes resultedin a mean flow score of 2.7 in the three animals at the thirty-minuteangiogram, which persisted out to the four-hour timepoint. In contrast,the delivery of 30 mg of NAT by pulsed infusion over sixty minutes wasfar less impressive. Although these data have not been statisticallycompared, the results appear to favor delivery of NAT as a more rapidpulse regimen.

EXAMPLE 4 Optimization of Pulse Volume

The pulse-spray infusion device is programmable for delivering pulsevolumes of 0.1 to 0.5 mL per pulse. To determine if the pulse volume hadany effect on the angiographic outcomes in the pig model, pulse volumesof 0.2 mL were compared to pulse volumes of 0.4 mL. NAT was delivered ata fixed dose of 10 mg (equivalent to 0.15 mg/kg in a 75 kg pig). Theresults are shown in Table 3. TABLE 3 Comparison of Angiographic ResultsUsing NAT Delivered in Pulse Volumes of 0.2 mL or 0.4 mL DELIV TIME(pulse ANGIOGRAPHIC FLOW SCORE DRUG DOSE CATH volume) 0 30 m 1 hr 2 hr 4hr NAT 10 mg PS 5 min 0.0 ± 1.0 ± 1.1 ± 1.6 ± 1.4 ± n = 7 2.5 (0.2 mL0.0 0.6 0.7 1.0 1.1 mg/mL q 15 sec) NAT 10 mg PS 5 min 0.0 ± 1.2 ± 1.0 ±0.8 ± 1.0 ± n = 6 2.5 (0.4 mL 0.0 0.4 0.6 0.8 0.6 mg/mL q 30 sec)

As shown in Table 3, at thirty minutes the mean angiographic score wasslightly higher in the 0.4 mL pulse volume group. However, at thefour-hour time point, the group mean was slightly higher in the 0.2 mLpulse volume. As such, no conclusion can be drawn from these studieswith regard to one pulse volume being superior to another.

The results in preceding text and tables suggest that virtually all ofthese NAT treatment regimens are effective in treating peripheral arteryocclusions with the method of delivery of this invention, and that theseresults are at least comparable to treatment with plasminogenactivators, such as urokinase, which is the current treatment of choicewith thrombolytic agents.

The results demonstrate that the PRO catheter with pulse-spray deliveryappears to provide superior angiographic results. Given the body weightof animals in these studies (70-100 kg), the fixed dosage of 30 mg isroughly equivalent to 0.3-0.4 mg/kg on a weight-adjusted basis.

Lowering the fixed dosage NAT to 10 mg resulted in a reduction of groupmean angiographic scores at four hours and in some animals, patency wasnot achieved. This indicates that a dose of 10 mg of NAT appears to be athreshold dose for biologic activity in this model. Given the bodyweight of animals in these studies (70-100 kg), the fixed dosage of 10mg is roughly equivalent to 0.1-0.15 mg/kg on a weight-adjusted basis.Varying the pulse volume from 0.2 mL to 0.4 mL did not appear toprofoundly impact the angiographic patency scores.

EXAMPLE 5 Establishment of Safe, Well Tolerated, Biologically EffectiveDose Range in Humans

No satisfactory literature exists on the serum concentration orbiochemical activity of α₂-macroglobulin in elderly patients withperipheral vascular disease (PVD). As α₂-macroglobulin concentrationsare a key determinant of the safety and likely to be related thetolerabilty of fibrinolytic metalloproteinases in vivo, across-sectional epidemiological study was conducted in patients with PVDto evaluate serum α₂-macroglobulin concentration and the fibrinolyticmetalloproteinase binding capacity (using NAT as the test agent).

Two hundred and sixteen patients were enrolled at two centers (ClevelandClinic Foundation, Cleveland, Ohio, and Rochester General Hospital,Rochester, N.Y.). Demographic information and other patientcharacteristics were collected and serum was obtained for measuringα₂-macroglobulin, the NAT binding capacity (by titration of individualpatient serum samples using an HPLC assay that detects unbound NAT) andother serum chemistry parameters. The primary endpoint was thedetermination of the relationship between the serum concentration ofα₂-macroglobulin and the amount of NAT (in micrograms per milliliter ofserum) that could be neutralized in vitro (NAT binding capacity).

A comparison of patient characteristics in this study with those of thetwo largest published studies of thrombolysis in PAO (i.e., the STILEand TOPAS studies) indicated that the patient population in this studywas representative of previous studies of thrombolysis in PAO; Forfurther details on the previous studies, see Annals of Surgery, Volume220, pages 251-266 (1994) and Ouriel et al., New England Journal ofMedicine, Volume 338, pages 1105-1111 (1998), respectively.

The estimated maximum dose (EMD) for NAT was calculated for each patientusing the NAT binding capacity and an estimate of each patient's plasmavolume. The study results predict that the average patient could receivea dosage of 1.7 mg/kg (delivered either locally or systemically) withoutexceeding the capacity of α₂-macroglobulin to bind and neutralize NAT.The results from the study are summarized in FIG. 4.

In FIG. 4, the estimated maximum dose of NAT was calculated for each ofthe 216 subjects in the study. The results for the study are depicted asa histogram, above, where a bell-shaped distribution can be observed byvisual inspection. The average patient in this study is predicted to becapable of tolerating 1.7 mg/kg of NAT (the peak of the bell-shapeddistribution). Dosages administered in animal studies are shown forreference (right hand side) and can be seen to be in excess of theestimated maximum dose of NAT for 99% of the study population.

Thus, the prescribed range of 0.025 to 1.7 mg/kg for the inventionrepresents a rational estimate of the dose patients can safely receive(based on plasma volume and NAT binding capacity for α₂-macroglobulin)without the appearance of free NAT in the circulation.

In conclusion, the results from the exemplified pharmacology studies,Examples 1-4, above, indicate the biological effectiveness of afibrinolytic metalloproteinase as a clot lysing agent in an animal modelof thrombosis where the thrombus is comparable in size and age to thatfrequently encountered in peripheral arterial occlusion in humans. Thedosages identified in the animal models were obtained without regard foror assessment of the potential toxicities in the animal. The effectivedosages in rabbits and dogs (3.7 and 4.0 mg/kg, respectively) asdescribed by Ahmed et al. and Markland et al. (above) might enable aveterinary use for fibrinolytic metalloproteinases. However, whenconsidered in the presence of human data, the administration of doses of3.7 and 4.0 mg/kg would have overdosed 99 percent of the studypopulation. Therefore, the published animal studies do not enable thetherapeutic use of fibrinolytic metalloproteinases in humans in a mannerthat is safe as well as biologically effective. The data presented inExample 5, on the other hand, do enable such use in humans.

1. A method for restoring patency to an indwelling vascular access device comprising contacting said vascular access device with a fibrinolytic metalloproteinase selected from the group consisting of fibrolase and Novel Acting Thrombolytic (NAT), in an amount sufficient to lyse a blood clot in, on, or attached to the indwelling vascular access device.
 2. The method of claim 1, wherein the fibrinolytic metalloproteinase is provided at a concentration ranging from 0.1 to 80 mg/ml.
 3. The method of claim 1, wherein the fibrinolytic metalloproteinase is provided at a concentration ranging from 0.1 to 50 mg/ml.
 4. The method of claim 1, wherein said vascular access device is a catheter device.
 5. The method of claim 1, wherein said vascular access device is a shunt.
 6. The method of claim 1, wherein said vascular access device is an access graft.
 7. The method of claim 1, wherein said vascular access device is a needle.
 8. The method of claim 1, wherein said fibrinolytic metalloproteinase is NAT.
 9. The method of claim 8, wherein said NAT comprises the amino acid sequence of SEQ ID NO:1.
 10. The method of claim 1, wherein said fibrinolytic metalloproteinase is a fibrolase.
 11. The method of claim 10, wherein said fibrolase comprises the amino acid sequence of SEQ ID NO:3.
 12. A method for restoring patency to a catheter device present in a human subject, said method comprising contacting said catheter device with Novel Acting Thrombolytic (NAT) comprising the amino acid sequence of SEQ ID NO: 1, wherein said contacting is done using a concentration of NAT ranging from 0.1 to 80 mg/ml.
 13. The method of claim 14, wherein the fibrinolytic metalloproteinase is provided at a concentration ranging from 0.1 to 50 mg/ml.
 14. A method for restoring function to an indwelling vascular access device comprising contacting said vascular access device with a fibrinolytic metalloproteinase selected from the group consisting of fibrolase and Novel Acting Thrombolytic (NAT), in an amount sufficient to lyse a blood clot in, on, or attached to the indwelling vascular access device.
 15. The method of claim 14, wherein the fibrinolytic metalloproteinase is provided at a concentration ranging from 0.1 to 80 mg/ml.
 16. The method of claim 14, wherein the fibrinolytic metalloproteinase is provided at a concentration ranging from 0.1 to 50 mg/ml.
 17. The method of claim 14, wherein said vascular access device is a catheter device.
 18. The method of claim 14, wherein said vascular access device is a shunt.
 19. The method of claim 14, wherein said vascular access device is an access graft.
 20. The method of claim 14, wherein said vascular access device is a needle.
 21. The method of claim 14, wherein said fibrinolytic metalloproteinase is NAT.
 22. The method of claim 21, wherein said NAT comprises the amino acid sequence of SEQ ID NO:1.
 23. The method of claim 14, wherein said fibrinolytic metalloproteinase is a fibrolase.
 24. The method of claim 23, wherein said fibrolase comprises the amino acid sequence of SEQ ID NO:3.
 25. A method for restoring function to a catheter device present in a human subject, said method comprising contacting said catheter device with Novel Acting Thrombolytic (NAT) comprising the amino acid sequence of SEQ ID NO:1, wherein said contacting is done using a concentration of NAT ranging from 0.1 to 80 mg/ml.
 26. The method of claim 14, wherein the fibrinolytic metalloproteinase is provided at a concentration ranging from 0.1 to 50 mg/ml.
 27. A method of lysing a clot associated with a medical device implanted in a human subject, said method comprising contacting said medical device with a fibrinolytic metalloproteinase selected from the group consisting of fibrolase and Novel Acting Thrombolytic (NAT), in an amount sufficient to lyse a blood clot in, on, or attached to the medical device.
 28. The method of claim 27, wherein the fibrinolytic metalloproteinase is provided at a concentration ranging from 0.1 to 80 mg/ml.
 29. The method of claim 27, wherein the fibrinolytic metalloproteinase is provided at a concentration ranging from 0.1 to 50 mg/ml.
 30. The method of claim 27, wherein said medical device is an indwelling catheter device.
 31. The method of claim 27, wherein said medical device is a hemodialysis access graft.
 32. The method of claim 27, wherein said fibrinolytic metalloproteinase is NAT.
 33. The method of claim 32, wherein said NAT comprises the amino acid sequence of SEQ ID NO:1.
 34. The method of claim 27, wherein said fibrinolytic metalloproteinase is a fibrolase.
 35. The method of claim 34, wherein said fibrolase comprises the amino acid sequence of SEQ ID NO:3.
 36. A method of lysing a clot associated with a medical device implanted in a human subject, said method comprising contacting said medical device with Novel Acting Thrombolytic (NAT) comprising the amino acid sequence of SEQ ID NO:1, wherein said contacting is done using a concentration of NAT ranging from 0.1 to 80 mg/ml.
 37. The method of claim 36, wherein the fibrinolytic metalloproteinase is provided at a concentration ranging from 0.1 to 50 mg/ml. 